Herpes simplex virus type 1 (HSV-1) is a significant human pathogen causing diseases such as mucocutaneous ulcers, keratitis, and encephalitis. In the USA, HSV-1 is the leading cause of blindness due to infection and the leading cause of sporadic encephalitis. Strains of HSV-1 display virulence patterns ranging from no disease to lethal encephalitis. The severity of keratitis also varies widely between viral strains. To date, little is known about role most HSV-1 genes play in the severity of an infection. It is likely that virulence differences are due to effects of multiple genes and the combination of alleles carried by a given strain of virus. Traditional methods for studying the effect of viral genes on virulence are not capable of identifying the effects of multiple genes. We are capable of generating recombinant HSV-1 strains by carrying out mixed infections between two strains and have found that these recombinants can vary widely in their virulence phenotypes. The overall goal of this study is to combine our ability to isolate recombinant viruses with varying disease phenotypes with our experience using next generation sequencing methods and novel statistical analysis of SNPs between the parental strains to identify virulence factors critical for ocular HSV-1 infection and encephalitis. This highly novel approach will allow us to identify the contribution of multiple genes in virulence and potentially identify a role for epistasis in determining the virulence phenotype of a given virus.